This competitive renewal application requests 5 years of support to investigate the biochemistry and physiology of homocysteine metabolism and its control by cystathionine beta synthase (CBS). The applicant has previously developed a functional assay for human CBS by complementation of CBS-deficient yeast for the ability to grown on cysteine-deficient medium. In the previous funding cycle, experiments based on the yeast assay led to the unexpected finding that the carboxyl-terminal domain of CBS is a negative regulator of catalytic activity carried out by the amino-terminal domain. Previous work had implicated the carboxy-terminal domain as being required for allosteric control of CBS activity by the physiologic positive regulator S-adenosyl methionine (SAM). The current application proposes to build upon these observations. In Aim 1, a structure-function analysis of the carboxy-terminal domain will be carried out to identify amino acid substitutions that will either enhance or suppress the ability of the carboxy-terminal domain to inhibit amino terminal enzymatic activity as inferred from the yeast complementation assay. The physiologic consequences of a mutation that suppresses inhibition (and therefore activates enzymatic activity) will be investigated in Aim 2 by constructing transgenic mice that express variant CBS proteins in a knockout background. In Aim 3, the ability of small molecules to effect changes in regulatory activity of the carboxy-terminal domain will be investigated by screening peptide expression libraries in yeast. Finally, Aim 4 will examine the potential relevance of these findings to allosteric control by AdoMet by determining the AdoMet binding site using a derivatized form of AdoMet that can be covalently cross-linked after photo-activation.